Fusion Partner Toolchest for the Stabilization and Crystallization of G Protein-Coupled Receptors

• Published in: Structure (London) Vol. 20; no. 6; pp. 967 - 976
• Main Authors: Chun, Eugene, Thompson, Aaron A., Liu, Wei, Roth, Christopher B., Griffith, Mark T., Katritch, Vsevolod, Kunken, Joshua, Xu, Fei, Cherezov, Vadim, Hanson, Michael A., Stevens, Raymond C.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 06-06-2012
• Subjects: Amino Acid Sequence; Animals; Cell Line; Chromatography, Gel; Cloning, Molecular; Crystallization; Crystallography, X-Ray; Cytochromes b - biosynthesis; Cytochromes b - chemistry; Cytochromes b - isolation & purification; Displays; Human; Humans; Molecular Sequence Data; Muramidase - biosynthesis; Muramidase - chemistry; Muramidase - isolation & purification; Protein Stability; Proteins; Receptor, Adenosine A2A - biosynthesis; Receptor, Adenosine A2A - chemistry; Receptor, Adenosine A2A - isolation & purification; Receptors; Receptors, G-Protein-Coupled - biosynthesis; Receptors, G-Protein-Coupled - chemistry; Receptors, G-Protein-Coupled - isolation & purification; Recombinant Fusion Proteins - biosynthesis; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - isolation & purification; Stability; Stabilization
• ISSN: 0969-2126; 1878-4186
• DOI: 10.1016/j.str.2012.04.010

Structural studies of human G protein-coupled receptors (GPCRs) have recently been accelerated through the use of a fusion partner that was inserted into the third intracellular loop. Using chimeras of the human β2-adrenergic and human A2A adenosine receptors, we present the methodology and data for the initial selection of an expanded set of fusion partners for crystallizing GPCRs. In particular, use of the thermostabilized apocytochrome b562RIL as a fusion partner displays certain advantages over previously utilized fusion proteins, resulting in a significant improvement in stability and structure of GPCR-fusion constructs.
► A method was developed for the selection of fusion domains for GPCR crystallization ► Apocytochrome b562RIL has advantages over previously utilized T4 lysozyme ► Diffraction quality crystals of two engineered GPCRs were successfully grown ► The method led to the crystal structure of the A2A adenosine receptor at 1.8 Å